Conformational regulation of Escherichia coli DNA polymerase V by RecA and ATP
Conformational regulation of Escherichia coli DNA polymerase V by RecA and ATP
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DOI：
10.1371/journal.pgen.1007956
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发表时间：
2019-02-01
期刊：
影响因子：
4.5
通讯作者：
Goodman, Myron F.
中科院分区：
文献类型：
--
作者：
Jaszczur, Malgorzata M.;Vo, Dan D.;Goodman, Myron F.
致突变跨损伤DNA聚合酶V（UmuD（2）C）作为DNA损伤诱导的SOS反应的一部分在大肠杆菌中被诱导，并受到多水平的调控。UmuC亚基被隔离在细胞膜上（空间调节），并在形成UmuD（2）C复合物后进入胞质溶胶，类似于SOS诱导后45分钟（时间调节）。然而，DNA结合和合成不能发生，直到pol V与RecA核蛋白丝（RecA*）和ATP相互作用以形成突变体复合物，pol V Mut = UmuD（2）C-RecA-ATP。RecA相对于UmuC的位置决定了pol V Mut是催化开启还是关闭（构象调节）。在这里，我们提出了三个相互关联的实验来解决构象调节的生化基础。我们首先研究了DNA合成过程中的动态失活和DNA合成不存在时的静态失活。单分子（SM）TIRF-FRET显微镜，然后用于探索多个方面的pol V Mut动力学。ATP/ATPS的结合触发构象转换，其相对于UmuC重定向RecA以激活pol V Mut。这个过程是聚合酶-DNA结合和合成所必需的。动态和静态失活过程都受温度和时间的控制，其中开关在37 ℃下快速（类似于1至1.5小时），在30 ℃下缓慢（类似于3至4小时），并且不需要ATP水解。Pol V Mut保留RecA处于激活和失活状态，但仅在激活时才与引物-模板（p/t）DNA结合。用两种形式的聚合酶pol V Mut-RecA wt和组成型诱导和超诱变性pol V Mut-RecA E38 K/C17进行研究。我们讨论了pol V Mut的构象调控，从体外生化分析确定，在RecA野生型和SOS组成的遗传背景中的pol V Mut在vivo.Author总结大肠杆菌上调超过40个基因的性质作为DNA损伤诱导的SOS调节子的一部分，其中许多参与DNA修复和细胞分裂。然而，三种DNA聚合酶，pols V，II和IV，也被诱导以拯救在持续模板病变处被阻断的复制叉。由UV诱变基因（umuDC）编码的Pol V（UmuD（2）C）主要负责UV诱导的染色体诱变的增加。然而，pol V是催化惰性的。需要与RecA核蛋白丝（RecA*）和ATP相互作用以将pol V转化为活化的突变体复合物，pol V Mut = UmuD（2）C-RecA-ATP。在这里，我们表明，聚合物V Mut在DNA合成过程中动态失活，静态合成的情况下。激活和失活状态是由构象开关，重新定位RecA相对于UmuC。转换速率在37 ℃比在30 ℃更快，并且不需要ATP水解。ATP（ATPS）结合起两个所需的调节作用：1）它允许pol V Mut与引物-模板DNA结合; 2）它触发RecA-UmuC构象转换，从而激活pol V Mut。
Mutagenic translesion DNA polymerase V (UmuD(2)C) is induced as part of the DNA damage-induced SOS response in Escherichia coli, and is subjected to multiple levels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD(2)C complex, similar to 45 min post-SOS induction (temporal regulation). However, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD(2)C-RecA-ATP. The location of RecA relative to UmuC determines whether pol V Mut is catalytically on or off (conformational regulation). Here, we present three interrelated experiments to address the biochemical basis of conformational regulation. We first investigate dynamic deactivation during DNA synthesis and static deactivation in the absence of DNA synthesis. Single-molecule (sm) TIRF-FRET microscopy is then used to explore multiple aspects of pol V Mut dynamics. Binding of ATP/ATPS triggers a conformational switch that reorients RecA relative to UmuC to activate pol V Mut. This process is required for polymerase-DNA binding and synthesis. Both dynamic and static deactivation processes are governed by temperature and time, in which on off switching is rapid at 37 degrees C (similar to 1 to 1.5 h), slow at 30 degrees C (similar to 3 to 4 h) and does not require ATP hydrolysis. Pol V Mut retains RecA in activated and deactivated states, but binding to primer-template (p/t) DNA occurs only when activated. Studies are performed with two forms of the polymerase, pol V Mut-RecA wt, and the constitutively induced and hypermutagenic pol V Mut-RecA E38K/C17. We discuss conformational regulation of pol V Mut, determined from biochemical analysis in vitro, in relation to the properties of pol V Mut in RecA wild-type and SOS constitutive genetic backgrounds in vivo.Author summary Escherichia coli upregulates more than 40 genes as part of the DNA damage-induced SOS regulon, many of which are involved in DNA repair and cell division. However, three DNA polymerases, pols V, II, and IV, are also induced to rescue replication forks blocked at persisting template lesions. Pol V (UmuD(2)C), encoded by the UV mutagenesis genes (umuDC), is primarily responsible for the increase in UV-induced chromosomal mutagenesis. However, pol V is catalytically inert. Interaction with a RecA nucleoprotein filament (RecA*) and ATP is required to convert pol V to an activated mutasome complex, pol V Mut = UmuD(2)C-RecA-ATP. Here, we show that pol V Mut deactivates dynamically during DNA synthesis, and statically in the absence of synthesis. Activated and deactivated states are governed by a conformational switch that repositions RecA relative to UmuC. Switching rates are more rapid at 37 than at 30 degrees C, and do not require ATP hydrolysis. ATP (ATPS) binding plays two required regulatory roles: 1) it allows binding of pol V Mut to primer-template DNA; 2) it triggers the RecA-UmuC conformational switch that activates pol V Mut.